Drug-Conjugates With a Targeting Molecule and Two Different Drugs

ABSTRACT

There is disclosed an improved ADC (antibody drug conjugate) type composition having at least two different drug payloads conjugated to a single targeting protein. More specifically, the present disclosure attaches a first drug conjugate to a dual Cysteine residue on a targeting protein and a second drug conjugate with a different drug to a Lys residue on the targeting protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/515,352, filed Oct. 15, 2014, which claims priority from U.S.Provisional Application No. 61/891,310 filed Oct. 15, 2013, the contentsof each of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present disclosure provides an improved ADC (antibody drugconjugate) type composition having at least two different drug payloadsconjugated to a single targeting protein. More specifically, the presentdisclosure attaches a first drug conjugate to a dual Cysteine residue ona targeting protein and a second drug conjugate with a different drug toa Lys residue on the targeting protein.

BACKGROUND

An antibody (or antibody fragment) can be linked to a payload drug toform an immunoconjugate that has been termed antibody-drug conjugate, orADC. The antibody causes the ADC to bind to the target cells. Often theADC is then internalized by the cell and the drug is released to treatthe cell. Because of the targeting, the side effects may be lower thanthe side effects of systemically administering the drug.

SUMMARY

The present disclosure provides active agent-conjugates that include atleast two different types of drugs. A first drug is conjugated tosulfhydryl groups of a targeting protein on Cys residues within fouramino acids of each other, such as on an antibody hinge region, and asecond drug conjugated to an amino groups of Lys side chains of thetargeting protein.

Specifically, the present disclosure provides a dual activeagent-conjugate having the structure of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:A is a targeting moiety;each D¹ is independently selected, where each D¹ is an active agent;each L¹ is independently a linker including at least one N (nitrogen)atom;each D² is independently selected, where each D² includes an activeagent;each L² is independently a linker;the E-component is an optionally substituted heteroaryl or an optionallysubstituted heterocyclyl;each L³ is an optionally substituted C₁-C₆ alkyl, or L³ may be null,when L³ is null the sulfur is directly connected to the E-component; andeach L⁴ is an optionally substituted C₁-C₆ alkyl, or L⁴ is null, when L⁴is null the sulfur is directly connected to the E-component;m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; andn is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

Preferably, the E-component includes a fragment selected from the groupconsisting of:

L³ is —(CH₂)—; and L⁴ is —(CH₂)—. L³ is null; and L⁴ is null.

L¹ includes —(CH₂)_(n)— where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.In some embodiments, L¹ includes —(CH₂CH₂O)_(n)— where n may be 1, 2, 3,4, 5, 6, 7, 8, 9, or 10. In some embodiments, L¹ includes Val-Cit-PAB,Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB,D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, or PAB. In someembodiments, L¹ includes peptide, oligosaccharide, —(CH₂)_(n)—,—(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB,PAB, or combinations thereof. Preferably, L¹ is selected from the groupconsisting of —(CH₂)_(n)—, —(CH₂CH₂O)_(n)— wherein n is an integer from1-10, a peptide,

wherein X¹ is N (nitrogen) or CH; Y¹ is N (nitrogen), or CH; and p is 0,1, or 2,

L² includes —(CH₂)_(n)— where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.In some embodiments, L² includes —(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4,5, 6, 7, 8, 9, or 10. In some embodiments, L² includes Val-Cit-PAB,Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB,D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, or PAB. In someembodiments, L² includes peptide, oligosaccharide, —(CH₂)_(n)—,—(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB,PAB, or combinations thereof. In some embodiments, L² includes anoncleavable unit. In some embodiments, the noncleavable unit includes—(CH₂)_(n)— where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, the noncleavable unit includes —(CH₂CH₂O)_(n)— where n maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L² includes acleavable unit. Preferably, the cleavable unit comprises a peptide.

The A component is an antibody (mAB) or fragment thereof. Alternatively,the A component comprises a Cys engineered antibody. In someembodiments, the A component comprises an antibody of which at least onepair of the interheavy chain disulfide bond was eliminated. In someembodiments, the A component comprises at least one modified L-Alanineresidue. In some embodiments, the A component comprises at least twomodified L-Alanine residues. In some embodiments, at least one L²includes —(CH₂)_(n)— where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Insome embodiments, at least one L² includes —(CH₂CH₂O)_(n)— where n maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, at least oneL² includes Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB,or PAB. In some embodiments, at least one L² includes a peptide, anoligosaccharide, —(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, PAB or combinations thereof.

Alternatively, the A component comprises at least two modified L-Alanineresidues. In some embodiments, at least one L² includes —(CH₂)_(n)—where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, atleast one L² includes —(CH₂CH₂O)_(n)— where n may be 1, 2, 3, 4, 5, 6,7, 8, 9, or 10. In some embodiments, at least one L² includesVal-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB,D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, or PAB. In someembodiments, at least one L² includes a peptide, an oligosaccharide,—(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB,Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg,Ala-Ala-Asn-PAB, Ala-PAB, PAB or combinations thereof.

A comprises at least one modified n-butyl L-a-amino acid. In someembodiments, A comprises at least one modified L-Lysine residue is froman L-Lysine residue of a peptide before conjugation. In someembodiments, A-NH together comprise at least one modified L-Lysineresidue. In some embodiments, the terminal nitrogen of the side chain ofan L-Lysine residue of a peptide before conjugation provides the NH ofA-NH. In some embodiments, A comprises the —(CH₂)₄— of the side chain ofan L-Lysine residue of a peptide before conjugation that provides the atleast one A-NH. In some embodiments, A comprises a modified n-butyla-amino acid residue.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows anti-Her-2 (A) dual conjugates (K-lock+C-lock) inducesenhanced antiproliferative effect in breast cancer cell lines, comparedto either single K-lock or C-lock conjugates. A, SKBR-3 (HER2 3+), B,HCC1954 (HER2 3+), C, MCF-7 (HER2+/−), were all treated with eithersingle conjugates or dual conjugates for 3 d. IC50 was determined forthe concentration that showed 50% inhibition of cell growth.

FIG. 2 shows anti-Her-2 (A) dual conjugates (K-lock+C-lock) inducesenhanced antiproliferative effect in breast cancer cell lines, comparedto either single K-lock or C-lock conjugates. A, SKBR-3 (HER2 3+), B,HCC1954 (HER2 3+), C, MCF-7 (HER2+/−), were all treated with eithersingle conjugates or dual conjugates for 3 days. Percentage of cellviability at above indicated concentrations were shown and compared withsingle conjugates.

FIG. 3 shows anti-Her-2 (A) dual conjugates (K-lock+C-lock) inducesenhanced antiproliferative effect in breast cancer cell lines,comparable to the combination of single K-lock and C-lock conjugates. A,SKBR-3 (HER2 3+), B, HCC1954 (HER2 3+), C, MCF-7 (HER2+/−), were alltreated with either single conjugates or dual conjugates for 3 d. IC50was determined as the concentration that showed 50% inhibition of cellgrowth.

DETAILED DESCRIPTION

The present disclosure provides a genus of dual-drug improved ADC's(antibody drug conjugates) that comprise two different drugs (D1 andD2), wherein the D1 conjugate is linked to a Lys residue of thetargeting protein (preferably an antibody or fragment thereof) that isalso called “K-Lock”, and the D2 or second drug conjugate is linked totwo nearby Cys residues on the targeting protein that is also called“C-Lock.” The present disclosure fulfills a long-felt need in the art tobe able to use a single targeting protein to deliver into a target cell(such as a cancer cell) two different drug payloads (D1 and D2).

Table 1 below shows structures of the K-lock conjugation and Table 2below shows structures for C-Lock conjugation. The present disclosure isbased on the ability to do both C-Lock and K-Lock with a singletargeting protein.

TABLE 1 Structures of K lock (Lys conjugation) compounds Compound nostructure 3

8

9

10

11

12

13

14

TABLE 2 Structures of C-lock (Cys conjugation) compounds Compound IDStructure 17

18

21

26

27

32

37

38

39

40

Table 3 below provides a list of those dual drug conjugates that areexemplified in this disclosure and shows both the D1 drug on the K-Lockside and the D2 drug on the C-Lock side.

TABLE 3 List of Dual conjugated (K lock and C lock) ADCs K lock (Lys) IDC lock (Cys) ID Dual conjugated ADC (Names used herein) 9 32 A*-9-32 918 A-9-18 9 38 A-9-38 9 40 A-9-40 3 17 A-3-17 3 40 A-3-40 3 37 A-3-37 1121 A-11-21 11 26 A-12-26 12 38 A-12-38 10 38 A-10-38 13 38 A-13-38 13 21A-13-21 8 21 A-8-21 10 21 A-10-21 14 21 A-14-21 8 26 A-8-26 14 21A-14-21 14 27 A-14-27 10 26 A-10-26 12 39 A-12-39 *A is an anti-HER2antibody Structure of the Dual conjugated ADC A-9-32

Structure of the Dual conjugated ADC A-9-18

Structure of the Dual conjugated ADC A-9-38

Structure of the Dual conjugated ADC A-9-40

Structure of the Dual conjugated ADC A-3-17

Structure of the Dual conjugated ADC A-3-40

Structure of the Dual conjugated ADC A-3-37

Structure of the Dual conjugated ADC A-11-21

Structure of the Dual conjugated ADC A-11-26

Structure of the Dual conjugated ADC A-12-38

Structure of the Dual conjugated ADC A-10-38

Structure of the Dual conjugated ADC A-13-38

Structure of the Dual conjugated ADC A-13-21

Structure of the Dual conjugated ADC A-8-21

Structure of the Dual conjugated ADC A-10-21

Structure of the Dual conjugated ADC A-14-21

Structure of the Dual conjugated ADC A-8-26

Structure of the Dual conjugated ADC A-14-26

Structure of the Dual conjugated ADC A-14-27

Structure of the Dual conjugated ADC A-10-26

Structure of the Dual conjugated ADC A-12-39

A targeting protein is conjugated to include at least two differenttypes of drugs by using two different conjugation methods, a firstconjugation method and a second conjugation method. The firstconjugation method to derivatize a polypeptide with a payload can beaccomplished using a maleimido or vinyl moiety which can react withindividual sulfhydryl group on an antibody via Michael additionreaction. A free sulfhydryl group can be formed by reducing a disulfidebond in an antibody. However, the structural integrity of the targetingprotein, such as an antibody, is compromised after opening disulfidebonds and attaching payloads to the exposed free thiols. Thecompositions and methods provided herein provide conjugation through acysteine residue without decreased structural stability. The secondconjugation method to derivatize a polypeptide with a payload isaccomplished by forming an amide bond with a lysine side chain. Due tothe presence of large number of lysine side chain amines with similarreactivity, this conjugation strategy can produce complex heterogeneousmixtures. The compositions and methods provided herein provideconjugation through lysine, where enhanced selectivity of the lysine canresult in a less heterogenous mixture. The conjugation methods aredesignated “first” and “second” for convenience of discussion and do notindicate the order of conjugation.

The term “pharmaceutically acceptable salt” are salts that retain thebiological effectiveness and properties of a compound and, which are notbiologically or otherwise undesirable for use in a pharmaceutical. Thedisclosed compounds are capable of forming acid and/or base salts byvirtue of the presence of amino and/or carboxyl groups or groups similarthereto. Pharmaceutically acceptable acid addition salts can be formedwith inorganic acids and organic acids. Inorganic acids from which saltscan be derived include, for example, hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organicacids from which salts can be derived include, for example, acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and thelike. Pharmaceutically acceptable base addition salts can be formed withinorganic and organic bases. Inorganic bases from which salts can bederived include, for example, sodium, potassium, lithium, ammonium,calcium, magnesium, iron, zinc, copper, manganese, aluminum, and thelike; particularly preferred are the ammonium, potassium, sodium,calcium and magnesium salts. Organic bases from which salts can bederived include, for example, primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, basic ion exchange resins, and the like, specificallysuch as isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, and ethanolamine. Many such salts are known in the art,as described in WO 87/05297, (incorporated by reference herein in itsentirety).

“C_(a) to C_(b)” or “C_(a-b)” in which “a” and “b” are integers refer tothe number of carbon atoms in the specified group. That is, the groupcan contain from “a” to “b”, inclusive, carbon atoms. Thus, for example,a “C₁ to C₄ alkyl” or “C₁₋₄ alkyl” group refers to all alkyl groupshaving from 1 to 4 carbons, that is, CH₃—, CH₃CH₂—, CH₃CH₂CH₂—,(CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and (CH₃)₃C—.

The term “halogen” or “halo” means fluorine, chlorine, bromine, oriodine.

An “alkyl” refers to a straight or branched hydrocarbon chain that isfully saturated. The alkyl group may have 1 to 20 carbon atoms (wheneverit appears herein, a numerical range such as “1 to 20” refers to eachinteger in the given range; e.g., “1 to 20 carbon atoms” means that thealkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbonatoms, etc., up to and including 20 carbon atoms, although the presentdefinition also covers the occurrence of the term “alkyl” where nonumerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 9 carbon atoms. The alkyl group could also be alower alkyl having 1 to 4 carbon atoms. The alkyl group may bedesignated as “C₁₋₄ alkyl” or similar designations. By way of example,“C₁₋₄ alkyl” indicates that there are one to four carbon atoms in thealkyl chain, that is, the alkyl chain is selected from the groupconsisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl, hexyl.

An “alkoxy” refers to the formula —OR wherein R is an alkyl as isdefined above, such as “C₁₋₉ alkoxy”, including but not limited tomethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy.

An “alkylthio” refers to the formula —SR wherein R is an alkyl as isdefined above, such as “C₁₋₉ alkylthio” and the like, including but notlimited to methylmercapto, ethylmercapto, n-propylmercapto,1-methylethylmercapto (isopropylmercapto), n-butylmercapto,iso-butylmercapto, sec-butylmercapto, tert-butylmercapto.

An “alkenyl” refers to a straight or branched hydrocarbon chaincontaining one or more double bonds. The alkenyl group may have 2 to 20carbon atoms, although the present definition also covers the occurrenceof the term “alkenyl” where no numerical range is designated. Thealkenyl group may also be a medium size alkenyl having 2 to 9 carbonatoms. The alkenyl group could also be a lower alkenyl having 2 to 4carbon atoms. The alkenyl group may be designated as “C₂₋₄ alkenyl” orsimilar designations. By way of example only, “C₂₋₄ alkenyl” indicatesthat there are two to four carbon atoms in the alkenyl chain, that is,the alkenyl chain is selected from the group consisting of ethenyl,propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl,buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl,1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl,buta-1,2,-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groupsinclude, but are in no way limited to, ethenyl, propenyl, butenyl,pentenyl, and hexenyl.

An “alkynyl” is a straight or branched hydrocarbon chain containing oneor more triple bonds. The alkynyl group may have 2 to 20 carbon atoms,although the present definition also covers the occurrence of the term“alkynyl” where no numerical range is designated. The alkynyl group mayalso be a medium size alkynyl having 2 to 9 carbon atoms. The alkynylgroup could also be a lower alkynyl having 2 to 4 carbon atoms. Thealkynyl group may be designated as “C₂₋₄ alkynyl” or similardesignations. By way of example, “C₂₋₄ alkynyl” indicates that there aretwo to four carbon atoms in the alkynyl chain, that is, the alkynylchain is selected from the group consisting of ethynyl, propyn-1-yl,propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Alkynylgroups include, for example, ethynyl, propynyl, butynyl, pentynyl, andhexynyl.

The term “aromatic” refers to a ring or ring system having a conjugatedpi electron system and includes both carbocyclic aromatic (e.g., phenyl)and heterocyclic aromatic groups (e.g., pyridine). The term includesmonocyclic or fused-ring polycyclic groups provided that the entire ringsystem is aromatic.

An “aryloxy” and “arylthio” refers to RO— and RS—, in which R is an arylas is defined above, such as “C₆₋₁₀ aryloxy” or “C₆₋₁₀ arylthio” such asphenyloxy.

An “aralkyl” or “arylalkyl” is an aryl group connected, as asubstituent, via an alkylene group, such as “C₇₋₁₄ aralkyl” and thelike, including but not limited to benzyl, 2-phenylethyl,3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group isa lower alkylene group (i.e., a C₁₋₄ alkylene group).

A “heteroaryl” refers to an aromatic ring or ring system that contain(s)one or more heteroatoms, that is, an element other than carbon,including but not limited to, nitrogen, oxygen and sulfur, in the ringbackbone. When the heteroaryl is a ring system, every ring in the systemis aromatic. The heteroaryl group may have 5-18 ring members (i.e., thenumber of atoms making up the ring backbone, including carbon atoms andheteroatoms), although the present definition also covers the occurrenceof the term “heteroaryl” where no numerical range is designated. In someembodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7ring members. The heteroaryl group may be designated as “5-7 memberedheteroaryl,” “5-10 membered heteroaryl,” or similar designations.Examples of heteroaryl rings include furyl, thienyl, phthalazinyl,pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl,pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinlinyl,benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, andbenzothienyl.

A “heteroaralkyl” or “heteroarylalkyl” is heteroaryl group connected, asa substituent, via an alkylene group. Examples include 2-thienylmethyl,3-thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl,isoxazollylalkyl, and imidazolylalkyl. In some cases, the alkylene groupis a lower alkylene group (i.e., a C₁₋₄ alkylene group).

A “carbocyclyl” means a non-aromatic cyclic ring or ring systemcontaining only carbon atoms in the ring system backbone. When thecarbocyclyl is a ring system, two or more rings may be joined togetherin a fused, bridged or spiro-connected fashion. Carbocyclyls may haveany degree of saturation provided that at least one ring in a ringsystem is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms, although the present definition also covers the occurrenceof the term “carbocyclyl” where no numerical range is designated. Thecarbocyclyl group may also be a medium size carbocyclyl having 3 to 10carbon atoms. The carbocyclyl group could also be a carbocyclyl having 3to 6 carbon atoms. The carbocyclyl group may be designated as “C₃-6carbocyclyl” or similar designations. Examples of carbocyclyl ringsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl,adamantyl, and spiro[4.4]nonanyl.

A “(carbocyclyl)alkyl” is a carbocyclyl group connected, as asubstituent, via an alkylene group, such as “C₄₋₁₀ (carbocyclyl)alkyl”including, cyclopropylmethyl, cyclobutylmethyl, cyclopropylethyl,cyclopropylbutyl, cyclobutylethyl, cyclopropylisopropyl,cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl,cycloheptylmethyl. In some cases, the alkylene group is a lower alkylenegroup.

“Cycloalkyl” means a fully saturated carbocyclyl ring or ring system.Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

“Cycloalkenyl” means a carbocyclyl ring or ring system having at leastone double bond, wherein no ring in the ring system is aromatic. Anexample is cyclohexenyl.

“Heterocyclyl” means a non-aromatic cyclic ring or ring systemcontaining at least one heteroatom in the ring backbone. Heterocyclylsmay be joined together in a fused, bridged or spiro-connected fashion.Heterocyclyls may have any degree of saturation provided that at leastone ring in the ring system is not aromatic. The heteroatom(s) may bepresent in either a non-aromatic or aromatic ring in the ring system.The heterocyclyl group may have 3 to 20 ring members (i.e., the numberof atoms making up the ring backbone, including carbon atoms andheteroatoms), although the present definition also covers the occurrenceof the term “heterocyclyl” where no numerical range is designated. Theheterocyclyl group may also be a medium size heterocyclyl having 3 to 10ring members. The heterocyclyl group could also be a heterocyclyl having3 to 6 ring members. The heterocyclyl group may be designated as “3-6membered heterocyclyl” or similar designations. In preferred sixmembered monocyclic heterocyclyls, the heteroatom(s) are selected fromone up to three of O (oxygen), N (nitrogen) or S (sulfur), and inpreferred five membered monocyclic heterocyclyls, the heteroatom(s) areselected from one or two heteroatoms selected from O (oxygen), N(nitrogen), or S (sulfur). Examples of heterocyclyl rings include,azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl,imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl,piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl,pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl,1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl,1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl,hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl,1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl,oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl,isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl,thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, andtetrahydroquinoline.

A “(heterocyclyl)alkyl” is a heterocyclyl group connected, as asubstituent, via an alkylene group. Examples include imidazolinylmethyland indolinylethyl.

An “acyl” is —C(═O)R, wherein R is hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl,and 5-10 membered heterocyclyl. Examples include formyl, acetyl,propanoyl, benzoyl, and acryl.

An “O-carboxy” group is a “—OC(═O)R” group in which R is selected fromhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl.

A “C-carboxy” group is a “—C(═O)OR” group in which R is selected fromhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein. A non-limiting example includes carboxyl (i.e.,—C(═O)OH).

A “cyano” group is a “—CN” group.

A “cyanato” group is an “—OCN” group.

An “isocyanato” group is a “—NCO” group.

A “thiocyanato” group is a “—SCN” group.

An “isothiocyanato” group is an “—NCS” group.

A “sulfinyl” group is an “—S(═O)R” group in which R is selected fromhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl.

A “sulfonyl” group is an “—SO₂R” group in which R is selected fromhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl.

An “S-sulfonamido” group is a “—SO₂NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

An “N-sulfonamido” group is a “—N(R_(A))SO₂R_(B)” group in which R_(A)and R_(b) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

An “O-carbamyl” group is a “—OC(═O)NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

An “N-carbamyl” group is an “—N(R_(A))C(═O)OR_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

An “O-thiocarbamyl” group is a “—OC(═S)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

A “urea” group is a “—N(R_(A))C(═O)NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

An “N-thiocarbamyl” group is an “—N(R_(A))C(═S)OR_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl.

A “C-amido” group is a “—C(═O)NR_(A)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

An “N-amido” group is a “—N(R_(A))C(═O)R_(B)” group in which R_(A) andR_(B) are each independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 memberedheteroaryl, and 5-10 membered heterocyclyl.

An “amino” group is a “—NR_(A)R_(B)” group in which R_(A) and R_(B) areeach independently selected from hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₃₋₇ carbocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl,and 5-10 membered heterocyclyl. An example is free amino (—NH₂).

An “aminoalkyl” group is an amino group connected via an alkylene group.

An “alkoxyalkyl” group is an alkoxy group connected via an alkylenegroup, such as a “C₂₋₈ alkoxyalkyl”.

A substituted group is derived from the unsubstituted parent group inwhich there has been an exchange of one or more hydrogen atoms foranother atom or group. Unless otherwise indicated, when a group isdeemed to be “substituted,” it is meant that the group is substitutedwith one or more substituent's independently selected from C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl (optionally substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆haloalkoxy), C₃-C₇-carbocyclyl-C₁-C₆-alkyl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heterocyclyl (optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), 5-10membered heterocyclyl-C₁-C₆-alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), aryl(optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆haloalkyl, and C₁-C₆ haloalkoxy), aryl(C₁-C₆)alkyl (optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, andC₁-C₆ haloalkoxy), 5-10 membered heteroaryl (optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy),5-10 membered heteroaryl(C₁-C₆)alkyl (optionally substituted with halo,C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, and C₁-C₆ haloalkoxy), halo,cyano, hydroxy, C₁-C₆ alkoxy, C₁-C₆ alkoxy(C₁-C₆)alkyl (i.e., ether),aryloxy, sulfhydryl (mercapto), halo(C₁-C₆)alkyl (e.g., —CF₃),halo(C₁-C₆)alkoxy (e.g., —OCF₃), C₁-C₆ alkylthio, arylthio, amino,amino(C₁-C₆)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, sulfinyl, sulfonyl, and oxo (═O). Wherever a group isdescribed as “optionally substituted” that group can be substituted withthe above substituents.

Wherever a substituent is depicted as a di-radical (i.e., has two pointsof attachment to the rest of the molecule), it is to be understood thatthe substituent can be attached in any directional configuration unlessotherwise indicated. For example, a substituent depicted as -AE- or

includes the substituent being oriented such that the A is attached atthe leftmost attachment point of the molecule as well as the case inwhich A is attached at the rightmost attachment point of the molecule.

“Subject” as used herein, means a human or a non-human mammal, e.g., adog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-humanprimate or a bird, e.g., a chicken, as well as any other vertebrate orinvertebrate.

Definitions

As used herein, common organic abbreviations are defined as follows:

Ac Acetyl

aq. AqueousBOC or Boc tert-ButoxycarbonylBrOP bromo tris(dimethylamino) phosphonium hexafluorophosphateBu n-Butyl° C. Temperature in degrees CentigradeDCM methylene chloride

DEPC Diethylcyanophosphonate

DIC diisopropylcarbodiimide

DIEA Diisopropylethylamine DMA N,N-Dimethylformamide DMFN,N-Dimethylformamide

EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

Et Ethyl

EtOAc Ethyl acetate

Eq Equivalents Fmoc 9-Fluorenylmethoxycarbonyl g Gram(s)

h Hour (hours)HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate

HOBT N-Hydroxybenzotriazole HOSu N-Hydroxysuccinimide

HPLC High-performance liquid chromatographyLC/MS Liquid chromatography-mass spectrometry

Me Methyl MeOH Methanol MeCN Acetonitrile mL Milliliter(s)

MS mass spectrometryPAB p-aminobenzylRP-HPLC reverse phase HPLCrt room temperaturet-Bu tert-Butyl

TEA Triethylamine

Tert, t tertiaryTFA Trifluoracetic acid

THF Tetrahydrofuran

TLC Thin-layer chromatography

μL Microliter(s)

A general synthesis procedure forms an activated ester (e.g. NHS) froman acid. For example, an acid was dissolved in DCM, and DMF was added toaid dissolution if necessary. N-hydroxysuccinimide (1.5 eq) was added,followed by EDC.HCl (1.5 eq). The reaction mixture was stirred at roomtemperature for 1 h until most of the acid was consumed. The progress ofthe reaction was monitored by RP-HPLC. The mixture was then diluted withDCM and washed successively with citric acid (aq. 10%) and brine. Theorganic layer was dried and concentrated to dryness. The crude productwas optionally purified by RP-HPLC or silica gel column chromatography.

Conjugation Methods, Spacers and Linkers Involved

Some embodiments provide a method of conjugating of a targeting moleculethrough a spacer or a multifunctional linker. In some embodiments, thespacer or multifunctional linker may include a 2- to 5-atom bridge. Insome embodiments, the method includes a single-step or sequentialconjugation approach. In some embodiments, the drug-conjugates include aspacer or a multifunctional linker. In some embodiments, the spacer ormultifunctional linker may include a noncleavable or cleavable unit suchas peptides.

Utilities and Applications

Some embodiments provide a method of treating a patient in need thereofcomprising administering an active agent-conjugate as disclosed anddescribed herein to said patient. In some embodiments, the patient mayhave cancer, immune diseases or diabetes.

Some embodiments provide a method of diagnosis or imaging comprisingadministering an active agent-conjugate as disclosed and describedherein to an individual.

Disclosed Compositions

The disclosed pharmaceutical compositions have a structure in Formula I

or a pharmaceutically acceptable salt thereof,wherein:A is a targeting protein;each D¹ is a first active agent;each L¹ is independently a linker including at least one N (nitrogen)atom;each D² is a second active agent;each L² is independently a linker;the E-component is an optionally substituted heteroaryl or an optionallysubstituted heterocyclyl;each L³ is an optionally substituted C₁-C₆ alkyl, or L³ may be null,when L³ is null the sulfur is directly connected to the E-component; andeach L⁴ is an optionally substituted C₁-C₆ alkyl, or L⁴ may be null,when L⁴ is null the sulfur is directly connected to the E-component;m and n are independently integers from 1-10.

Preferably, A is selected from the group consisting of a monoclonalantibody (mAB), and an antibody fragment.

D¹ and D² are different drug compounds, preferably an anti-cancer drugor an immune modulator. Examples of D1 and D2 are tubulin binders, DNAalkylating agents, HSP90 inhibitors, DNA topoisomerase inhibitors,anti-epigenetic agents, HDAC inhibitors, anti-metabolism agents,proteasome inhibitors, an siRNA, an antisense DNA, epothilone A,epothilone B, or paclitaxel.

L¹ may include a spacer or a multifunctional linker. L¹ may include aspacer and a multifunctional linker. In some embodiments, L¹ may includea multifunctional linker. In some embodiments, each L¹ may be a linker,wherein the linker may be cleavable or non-cleavable under biologicalconditions. In some embodiments, the linker may be cleavable by anenzyme. In some embodiments, L¹ may include Linker.

In some embodiments, L² may include a spacer or a multifunctionallinker. In some embodiments, L² may include a spacer and amultifunctional linker. In some embodiments, L² may include amultifunctional linker. In some embodiments, each L² may be a linker,wherein the linker may be cleavable or non-cleavable under biologicalconditions. In some embodiments, the linker may be cleavable by anenzyme. In some embodiments, L² may include Linker.

L² includes a cyclic group including at least one N (nitrogen) atom. Insome embodiments, L² includes a cyclic group including at least two N(nitrogen) atoms. In some embodiments, L² includes a cyclic groupincluding at least one N (nitrogen) atom and a spacer.

A comprises at least one modified L-Alanine residue. In someembodiments, A comprises at least two modified L-Alanine residues. Insome embodiments, A comprises at least one modified L-Alanine residuethat is connected to at least one sulfur. In some embodiments, at leastone modified L-Alanine residue is from an L-Cysteine residue of apeptide before conjugation.

A comprises at least one modified n-butyl L-a-amino acid. In someembodiments, A comprises at least one modified L-Lysine residue is froman L-Lysine residue of a peptide before conjugation. In someembodiments, A-NH together comprise at least one modified L-Lysineresidue. In some embodiments, the terminal nitrogen of the side chain ofan L-Lysine residue of a peptide before conjugation provides the NH ofA-NH of Formula I. In some embodiments, A comprises the —(CH₂)₄— of theside chain of an L-Lysine residue of a peptide before conjugation thatprovides the at least one A-NH of Formula I. In some embodiments, Acomprises a modified n-butyl a-amino acid residue.

Linker may be a peptide.

Linker may include an oligosaccharide. For example, Linker may includechitosan. In some embodiments, L² may include Linker and —(CH₂)_(n)—where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L² mayinclude Linker and —(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10.

Linker may include —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or10.

Linker may include —(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10.

Linker may include Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB,Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg,Ala-Ala-Asn-PAB, Ala-PAB, PAB, or the like.

Linker may include any combination of peptide, oligosaccharide,—(CH₂)_(n)—, —(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB,Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg,Ala-Ala-Asn-PAB, Ala-PAB, PAB, and the like.

A spacer is any combination of peptide, oligosaccharide, —(CH₂)_(n)—,—(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB,PAB.

includes a 4-carbon bridge.

L³, L⁴ and a portion of the E-component include a 4-carbon bridge.

includes,

The S-linked portion of

comprises a modified L-Alanine residue, wherein

connects to a sulfur of a reduced disulfide bond through a bridgecontaining 2 to 5 atoms. For example, the structure indicated by

includes a fragment selected from the group consisting of:

The S-linked (sulfur-linked) portion of

comprises a modified L-Alanine residue. In some embodiments, theS-linked (sulfur-linked) portion of

comprises a modified L-Alanine residue wherein the modified L-Alaninecomponent of

is from an L-Cysteine residue of a peptide before conjugation. Eachsulfur of

is from an L-Cysteine of a peptide before conjugation.

The structural component

is:

The E-component includes a fragment selected from the group consistingof:

L¹ may include,

The active agent may be selected from the group consisting of tubulinbinders, DNA alkylators, DNA intercalator, enzyme inhibitors, immunemodulators, peptides, and nucleotides.

At least one L¹ or L² includes —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6,7, 8, 9, or 10. In some embodiments, at least one L¹ or L² includes—(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, at least one L¹ or L² includes Val-Cit-PAB, Val-Ala-PAB,Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys,Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, or PAB. In some embodiments, atleast one L¹ or L² includes a peptide, an oligosaccharide, —(CH₂)_(n)—,—(CH₂CH₂O)_(n)—, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB,Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB,or PAB.

The targeting moiety may be an antibody. In some embodiments, thetargeting moiety may be a monoclonal antibody (mAb). The A componentcomprises a humanized antibody. In some embodiments, the A componentcomprises a chimeric antibody. In some embodiments, the A componentcomprises a bispecific antibody. In some embodiments, the targetingmoiety may be an antibody fragment, surrogate, or variant.

The targeting moiety may be HuM195-Ac-225, HuM195-Bi-213, Anyara(naptumomab estafenatox; ABR-217620), AS1409, Zevalin (ibritumomabtiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307, CR011-vcMMAE,Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242, IMGN388,IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(huI4.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L19-¹³¹I, L19-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.In some embodiments, the targeting moiety may comprise, consist of, orconsist essentially of the antibody portion of HuM195-Ac-225,HuM195-Bi-213, Anyara (naptumomab estafenatox; ABR-217620), AS1409,Zevalin (ibritumomab tiuxetan), BIIB015, BT-062, Neuradiab, CDX-1307,CR011-vcMMAE, Trastuzumab-DM1 (R3502), Bexxar (tositumomab), IMGN242,IMGN388, IMGN901, ¹³¹I-labetuzumab, IMMU-102 (⁹⁰Y-epratuzumab), IMMU-107(⁹⁰Y-clivatuzumab tetraxetan), MDX-1203, CAT-8015, EMD 273063(hu14.18-IL2), Tucotuzumab celmoleukin (EMD 273066; huKS-IL2),¹⁸⁸Re-PTI-6D2, Cotara, L19-IL2, Teleukin (F16-IL2), Tenarad (F16-¹³¹I),L19-¹³¹I, L19-TNF, PSMA-ADC, DI-Leu16-IL2, SAR3419, SGN-35, or CMC544.

The targeting moiety may be Brentuximab vedotin, Trastuzumab emtansine,Inotuzumab ozogamicin, Lorvotuzumab mertansine, Glembatumumab vedotin,SAR3419, Moxetumomab pasudotox, Moxetumomab pasudotox, AGS-16M8F,AGS-16M8F, BIIB-015, BT-062, IMGN-388, or IMGN-388.

The targeting moiety may comprise, consist of, or consist essentially ofthe antibody portion of Brentuximab vedotin, Trastuzumab emtansine,Inotuzumab ozogamicin, Lorvotuzumab mertansine, Glembatumumab vedotin,SAR3419, Moxetumomab pasudotox, Moxetumomab pasudotox, AGS-16M8F,AGS-16M8F, BIIB-015, BT-062, IMGN-388, or IMGN-388.

The targeting moiety may comprise, consist of, or consist essentially ofBrentuximab, Inotuzumab, Gemtuzumab, Milatuzumab, Trastuzumab,Glembatumomab, Lorvotuzumab, or Labestuzumab.

Conjugation Method I

Scheme I. G is selected from the group consisting of —F, —Cl, —Br, —I,—N₃, —OR, SR, —ONRR, RC(═O)O—, and RSO₂—O—; and R is optionallysubstituted alkyl, or optionally substituted aryl.

General Conjugation Procedure I-A:

To a solution of 0.5-50 mgs/mL of I-A in buffer at pH 6.0-9.0 with 0-30%organic solvent, is added 0.1-10 eq of activated carboxylic componentI-B in a manner of portion wise or continuous flow. The reaction isperformed at 0-40° C. for 0.5-50 hours with gentle stirring or shaking,monitored by HIC-HPLC. The resultant crude ADC product undergoesnecessary down-stream steps of desalt, buffet changes/formulation, andoptionally, purification, using the state-of-art procedures. The ADCproduct I-C is characterized by HIC-HPLC, SEC, RP-HPLC, and optionallyLC-MS.

Scheme I. X is selected from the group consisting of —Cl, —Br, —I, andRSO₂—O—; and R is optionally substituted alkyl, or optionallysubstituted aryl

General Conjugation Procedure I-B:

To the ADC product I-C, 0.5-50 mgs/mL, in a certain buffet at pH5.0-9.0, such as PBS, is added 0.5-100 eq of reducing agent such as TCEPand DTT to afford intermediate I-D. The reduction is performed at 0-40°C. for 0.5-40 hours with gentle stirring or shaking, and then thereducing agent is removed by column or ultrafiltration. To intermediateI-D, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0, such as PBS, with0-30% of organic co-solvent such as DMA, is added 0.5-10 eq of theactivated drug-linker reactant I-E. The reaction is conducted at 0-40°C. for 0.5-40 hours with gentle stirring or shaking, monitored byHIC-HPLC. The resultant crude ADC product I-E undergoes necessarydown-stream steps of desalt, buffet changes/formulation, and optionally,purification, using the state-of-art procedures. The final ADC productI-E is characterized by HIC-HPLC, SEC, RP-HPLC, and optionally LC-MS.

Conjugation Method II

General Conjugation Procedure II-A:

To a mixture of I-A, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0,such as PBS, is added 0.5-100 eq of reducing agent such as TCEP and DTTto afford intermediate II-A. The reduction is performed at 0-40° C. for0.5-40 hours with gentle stirring or shaking, and then the reducingagent is removed by column or ultrafiltration.

Scheme IV. X is selected from the group consisting of —Cl, —Br, —I, andRSO₂—O—; and R is optionally substituted alkyl, or optionallysubstituted aryl

General Conjugation Procedure II-B:

To intermediate II-A, 0.5-50 mgs/mL, in a certain buffet at pH 5.0-9.0,such as PBS, with 0-30% of organic co-solvent such as DMA, is added0.5-10 eq of the activated drug-linker reactant I-E. The reaction isconducted at 0-40° C. for 0.5-40 hours with gentle stirring or shaking,monitored by HIC-HPLC. The resultant crude ADC product II-B undergoesnecessary down-stream steps of desalt, buffet changes/formulation, andoptionally, purification, using the state-of-art procedures.

To a solution of 0.5-50 mgs/mL of II-B in buffer at pH 6.0-9.0 with0-30% organic solvent, is added 0.1-10 eq of activated carboxyliccomponent I-B in a manner of portion wise or continuous flow. Thereaction is performed at 0-40° C. for 0.5-50 hours with gentle stirringor shaking, monitored by HIC-HPLC. The resultant crude ADC product I-Fundergoes necessary down-stream steps of desalt, buffetchanges/formulation, and optionally, purification, using thestate-of-art procedures. The ADC product I-F is characterized byHIC-HPLC, SEC, RP-HPLC, and optionally LC-MS.

Examples of activated carboxylic component I-B include:

wherein G is selected from the group consisting of —F, —Cl, —Br, —I,—N₃, —OR, SR, —ONRR, RC(═O)O—, and RSO₂—O—; and R is optionallysubstituted alkyl, or optionally substituted aryl.

Examples of activated drug-linker reactant I-E are:

Examples of compounds of Formula I:

Example 1

This example illustrates the synthesis of compound 3.

To a solution of compound 1 (74 mg, 0.1 mmol) in THF (5 mL) was addedbromoacetic acid (70 mg, 5 eq.), followed by aq. saturated NaHCO₃ (2mL). The mixture was stirred at room temperature for 3 h and thenacidified with 1N hydrochloric acid. The mixture was extracted withethyl acetate and the organic layer was dried and concentrated. Thecrude product was purified by RP-HPLC to give compound 2 as a whitepowder after lyophilization (72 mg, 91%). MS m/z 795.5 [M+H]⁺.

Compound 2 (72 mg) was converted to its corresponding NHS ester (Generalprocedure). The NHS ester was dissolved in THF (2 mL). A solution of4-piperidine carboxylic acid (60 mg) in aq. saturated NaHCO₃ (1 mL) wasadded and the mixture was stirred at room temperature for 1 h. Themixture was then acidified with acetic acid and concentrated to dryness.The residue was purified by RP-HPLC to give compound 3 as a white powder(63 mg). MS m/z 906.6 [M+H]⁺.

Example 2

This example illustrates the synthesis of compound 8.

To a stirred solution of compound 4 (95 mg, 0.2 mmol) and compound 5(TFA salt, 146 mg, 0.2 mmol, prepared as described in WO 2013/173392) inDMF (4 mL) was added DIEA (0.14 mL), followed by HATU (80 mg). After 10min, piperidine (0.4 mL) was added to the reaction and the mixture wasstirred at room temperature for 30 min. The reaction mixture wasconcentrated and the residue was purified by RP-HPLC to give compound 6as TFA salt (141 mg, 73%). MS m/z 850.5 [M+H]⁺.

Compound 6 (141 mg) and 7 (37 mg) were dissolved in DMF (3 mL). DIEA(0.1 mL) was added, followed by HATU (57 mg). The reaction was stirredat room temperature for 30 min. 1N solution of aq. NaOH (2 mL) was addedand the reaction was stirred at room temperature for 2 h. Acetic acid(0.5 mL) was added to the reaction and the mixture was concentrated. Theresidue was purified by RP-HPLC to give compound 8 as a white powder(115 mg). MS m/z 1061.5 [M+H]+.

Example 3

TABLE 4 Compound ID Structure 9

10

11

12

13

14

Compounds 9, 10, 11, 12, 13, and 14 were prepared as described in WO2013/173392, the disclosure of which is incorporated by referenceherein.

Example 4

To a solution of compound 15 (0.1 mmol, prepared as described in WO2013/173392, the disclosure of which is incorporated by referenceherein) in THF (3 mL) was added a solution of compound 16 (0.15 mmol, 67mg) in acetonitrile/water (1/1, v/v, 1 mL), followed by DIEA (50 μL).After 30 min, the reaction was acidified and concentrated. The residuewas purified by reverse phase HPLC to give compound 17 as a white solid(87 mg). MS m/z 1243.6 [M+H]+.

Example 5

Compound 2 (0.1 mmol, 80 mg) and compound 16 (0.1 mmol, 45 mg) weredissolved in DCM/DMF (10/1, v/v, 3 mL). DIEA (20 μL) was added, followedby DIC (25 μL). The mixture was stirred at room temperature for 10 min.DCM was evaporated and the residue was purified by reverse phase HPLC togive compound 18 as a white powder (66 mg, 53%). MS m/z 1130.6 [M+H]+.

Example 6

Compound 19 (0.06 mmol, 36 mg) and compound 20 (0.05 mmol, 60 mg, TFAsalt) were dissolved in DCM/DMF (4/1, v/v, 3 mL). DIEA (25 μL) wasadded, followed by DIC (15 μL). The mixture was stirred at roomtemperature for 10 min. DCM was evaporated and the residue was purifiedby reverse phase HPLC to give compound 21 as a white powder (41 mg,49%). MS m/z 1572.8 [M+H]⁺.

Example 7

To a solution of compound 22 (54 mg, 0.1 mmol) in anhydrous DMF (3 mL)was added compound 23 (80 mg) and DIEA (20 μL). The mixture was stirredat room temperature for 2 h. Piperidine (40 μL) was added. After 3 h,the mixture was added dropwise to 100 mL of ether under vigorousstirring. The precipitated solid was collected and purified by reversephase HPLC to give compound 24 as a yellow solid (75 mg). MS m/z 947.3[M+H]⁺.

Compound 24 (75 mg) and compound 25 (42 mg) were dissolved in DCM/DMF(4/1, v/v, 3 mL). DIEA (20 μL) was added, followed by DIC (20 μL). Themixture was stirred at room temperature for 20 min. DCM was evaporatedand the residue was purified by reverse phase HPLC to give compound 26as a yellow powder (48 mg). MS m/z 1447.5 [M+H]⁺.

Example 8

Compound 27 was synthesized from Bleomycin using the same procedure asdescribed for compound 26. MS m/z 2320.8 [M+H]⁺.

Example 9

Preparation of compound 30: To a solution of compound 28 (97 mg, 0.125mmol) in 3 mL of DMF was added HATU (48 mg, 0.125 mmol), DIEA (52 mg,0.4 mmol), and compound 29 (100 mg, 0.125 mmol). After 1 h, to themixture was added piperidine (300 uL) and the mixture was stirred for 10min. Then the mixture was evaporated and purified by HPLC to givecompound 30 (83 mg, 50%). MS m/z 1224.5 (M+H).

Preparation of compound 32: To a solution of compound 30 (26 mg, 0.074mmol) in 1 mL of DCM was added DIC (46 mg, 0.037 mmol). After 10 min, asolution of compound 31 (41 mg, 0.031 mmol) and DIEA (17 μL) in 2 mL ofDCM was added and the mixture was stirred for 30 min. The solvent wasevaporated under vacuum and the residue was purified by HPLC to givecompound 32 (30 mg, 63%). MS m/z 1554.4 (M+H).

Example 10 Preparation of Compound 10:

Preparation of compound 35: To a solution of compound 33 (64 mg, 0.077mmol) in 3 mL of DMF was added 34 (97 mg, 0.077 mmol), HOBt (5 mg, 0.04mmol) and DIEA (13 mg, 0.1 mmol). After 24 h, reaction was done by HPLC,and 300 μL of piperidine was added. After 1 h, the mixture was purifiedby HPLC to give compound 35 (76 mg, 62%). MS m/z 1607.7 (M+H).

Preparation of compound 37: To a solution of compound 36 (31 mg, 0.09mmol) in 1 mL of DCM was added DIC (60 mg, 0.045 mmol). After 10 min, asolution of compound 35 (77 mg, 0.045 mmol) and DIEA (25 μL) in 2 mL ofDCM was added and the mixture was stirred for 30 min. The solvent wasevaporated under vacuum and the residue was purified by HPLC to givecompound 37 (60 mg, 69%). MS m/z 1930.6 (M+H).

Example 11

Compounds 38 and 39 were prepared as described in WO 2013/173391, thedisclosure of which is incorporated by reference herein.

Example 12

Compound 40 was synthesized from compound HTI-286 using the sameprocedure as described for compound 26. MS m/z 1366.7 [M+H]⁺.

Example 13

This example provides the results of EC50 assays of the designated dualdrug conjugated antibodies measured in vitro in specified cells.

TABLE 5.1 Dual Cytotoxic activity EC50 (nM) conjugated MDA- MDA- ADCSKBR-3 HCC1954 BT474 MB-175 SKOV_3 MCF-7 468 A-9-38 0.057 0.072 N/D N/DN/D >100 >100 A-3-17 N/D N/D 0.188 0.322 N/D 16.08 23.5 A-11-21 0.670.136 31.99 N/D 0.537 34.01 37.52 A-12-38 0.062 0.036 N/D N/DN/D >100 >100 A-10-38 0.081 0.1 N/D N/D N/D >100 >100 A-13-38 0.066 0.04N/D N/D N/D >100 >100 A-13-21 N/D N/D >100 N/D 0.4 N/D N/D A-8-21 0.120.1711 10.74 N/D 1.986 >100 48.61 A-10-21 0.03 0.039 N/D N/D N/D >10032.72 A-14-21 >100 0.509 N/D N/D N/D >100 >100 A-14-27 >100 >100 N/D N/DN/D >100 >100 A-12-39 0.028 0.024 0.132 N/D 0.326 5.816 >100 A-9 0.0410.138 0.423 3.635 0.405 >100 >100 A-3 0.055 0.16 >100 3.669 1.716 17.8424.85 A-11 0.046 0.04 0.219 N/D 0.518 14.3 6.486 A-12 0.047 0.03 0.2 N/D0.388 >100 >100 A-10 0.015 0.008 N/D N/D N/D >100 >100 A-13 0.028 0.0140.169 N/D 0.307 >100 >100 A-21 0.074 0.586 9.325 N/D 0.841 >100 53.53A-8 >100 >100 N/D N/D N/D >100 >100 A-38 0.626 0.81 N/D N/DN/D >100 >100 A-27 >100 >100 N/D N/D N/D >100 >100 A-39 0.038 0.0230.093 N/D 0.225 >100 >100 A-17 N/D N/D 0.153 0.186 N/D >100 >100

TABLE 5.2 Dual conjugated Cytotoxic activity EC50 (nM) ADC SKBR-3HCC1954 BT474 MDA-MB-175 SKOV_3 MCF-7 A-9-38 0.057 0.072 N/D N/DN/D >100 A-3-17 N/D N/D 0.188 0.322 N/D 16.08 A-11-21 0.67 0.136 31.99N/D 0.537 34.01 A-12-38 0.062 0.036 N/D N/D N/D >100 A-10-38 0.081 0.1N/D N/D N/D >100 A-13-38 0.066 0.04 N/D N/D N/D >100 A-13-21 N/DN/D >100 N/D 0.4 N/D A-8-21 0.12 0.1711 10.74 N/D 1.986 >100 A-10-210.03 0.039 N/D N/D N/D >100 A-14-21 >100 0.509 N/D N/D N/D >100A-14-27 >100 >100 N/D N/D N/D >100 A-12-39 0.028 0.024 0.132 N/D 0.3265.816 A-9 0.041 0.138 0.423 3.635 0.405 >100 A-3 0.055 0.16 >100 3.6691.716 17.84 A-11 0.046 0.04 0.219 N/D 0.518 14.3 A-12 0.047 0.03 0.2 N/D0.388 >100 A-10 0.015 0.008 N/D N/D N/D >100 A-13 0.028 0.014 0.169 N/D0.307 >100 A-21 0.074 0.586 9.325 N/D 0.841 >100 A-8 >100 >100 N/D N/DN/D >100 A-38 0.626 0.81 N/D N/D N/D >100 A-27 >100 >100 N/D N/DN/D >100 A-39 0.038 0.023 0.093 N/D 0.225 >100 A-17 N/D N/D 0.153 0.186N/D >100

TABLE 5.3 Dual conjugated Cytotoxic activity EC50 (nM) ADC SKBR-3HCC1954 BT474 MDA-MB-175 SKOV_3 MCF-7 A-9-38 0.057 0.072 N/D N/DN/D >100 A-3-17 N/D N/D 0.188 0.322 N/D 16.08 A-11-21 0.67 0.136 31.99N/D 0.537 34.01 A-12-38 0.062 0.036 N/D N/D N/D >100 A-10-38 0.081 0.1N/D N/D N/D >100 A-13-38 0.066 0.04 N/D N/D N/D >100 A-13-21 N/DN/D >100 N/D 0.4 N/D A-8-21 0.12 0.1711 10.74 N/D 1.986 >100 A-10-210.03 0.039 N/D N/D N/D >100 A-14-21 >100 0.509 N/D N/D N/D >100A-14-27 >100 >100 N/D N/D N/D >100 A-12-39 0.028 0.024 0.132 N/D 0.3265.816

TABLE 5.4 Dual Cytotoxic activity EC50 (nM) conjugated MDA- MDA- ADCSKBR-3 HCC1954 BT474 MB-175 SKOV_3 MCF-7 468 A-9-38 0.057 0.072 N/D N/DN/D >100 >100 A-3-17 N/D N/D 0.188 0.322 N/D 16.08 23.5 A-11-21 0.670.136 31.99 N/D 0.537 34.01 37.52 A-12-38 0.062 0.036 N/D N/DN/D >100 >100 A-10-38 0.081 0.1 N/D N/D N/D >100 >100 A-13-38 0.066 0.04N/D N/D N/D >100 >100 A-13-21 N/D N/D >100 N/D 0.4 N/D N/D A-8-21 0.120.1711 10.74 N/D 1.986 >100 48.61 A-10-21 0.03 0.039 N/D N/D N/D >10032.72 A-14-21 >100 0.509 N/D N/D N/D >100 >100 A-14-27 >100 >100 N/D N/DN/D >100 >100 A-12-39 0.028 0.024 0.132 N/D 0.326 5.816 >100

Example 14

This example provides a description of the comparative activity dataprovided in the Figures. On the first day, a specific tumor cell line,such as SKBR-3, was plated at 20-30% confluence in 100 μl culture mediumon a 96 well culture plate (Corning). The cells were incubated in a CO₂incubator at 37° C. overnight. On second day, a dual conjugate ADC, suchas A-3-17, was serially diluted to the culture medium at 19:60 ratio,with starting concentration of 100 nM. 5 μl of the serially dilutedconjugates were added to the 96 well plate containing SKRB-3 cells.SKBR-3 cells with dual conjugate ADC, A-3-17 were incubated at 37° C.for 72 hours. The viability of tumor cell treated with dual conjugateADC, A-3-17 was then measured using a cell viability kit, CelltitreGlo(Promega G-7573) according to the manufacturer protocol on a platereader (SpectraMax L from Molecular device). The IC₅₀ value, 50%inhibition of cell growth was calculated using a curve fitting software,Graphpad Prism.

FIG. 1 shows anti-Her-2 (A) dual conjugates (K-lock+C-lock) inducesenhanced antiproliferative effect in breast cancer cell lines, comparedto either single K-lock or C-lock conjugates. A, SKBR-3 (HER2 3+), B,HCC1954 (HER2 3+), C, MCF-7 (HER2+/−), were all treated with eithersingle conjugates or dual conjugates for 3 d. IC₅₀ was determined forthe concentration that showed 50% inhibition of cell growth.

FIG. 2 shows anti-Her-2 (A) dual conjugates (K-lock+C-lock) inducesenhanced antiproliferative effect in breast cancer cell lines, comparedto either single K-lock or C-lock conjugates. A, SKBR-3 (HER2 3+), B,HCC1954 (HER2 3+), C, MCF-7 (HER2+/−), were all treated with eithersingle conjugates or dual conjugates for 3 days. Percentage of cellviability at above indicated concentrations were shown and compared withsingle conjugates.

FIG. 3 shows anti-Her-2 (A) dual conjugates (K-lock+C-lock) inducesenhanced antiproliferative effect in breast cancer cell lines,comparable to the combination of single K-lock and C-lock conjugates. A,SKBR-3 (HER2 3+), B, HCC1954 (HER2 3+), C, MCF-7 (HER2+/−), were alltreated with either single conjugates or dual conjugates for 3 d. IC₅₀was determined as the concentration that showed 50% inhibition of cellgrowth.

1-15. (canceled)
 16. A method of preparing a dual-drug conjugate, themethod comprising conjugating a first moiety comprising D¹ to a lysineside chain of A and conjugating a second moiety comprising D² tocysteine side chains of A, thereby producing a dual-drug conjugatecomprising a structure of Formula I

or a pharmaceutically acceptable salt thereof, wherein: A comprises anantibody or antigen-binding antibody fragment; D¹ is an active agent;each L1 is independently a linker comprising at least one N (nitrogen)atom; the nitrogen between the antibody or antigen-binding antibodyfragment and the carbonyl of C(O)-L¹-D¹ is the nitrogen of a Lys sidechain of the antibody or antigen-binding antibody fragment; D² an activeagent, wherein D¹ and D² are different active agents; each L² isindependently a linker; the E-component is selected from the groupconsisting of

L³ is —(CH₂)—; L⁴ is —(CH₂)—; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; andn is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
 10. 17. The method of claim 16,wherein the first moiety is conjugated before conjugation of the secondmoiety.
 18. The method of claim 16, wherein the first moiety isconjugated after conjugation of the second moiety.
 19. The method ofclaim 16, wherein conjugating the first moiety to the lysine side chainof A comprises reacting a compound of formula I-A with a compound offormula I-B to provide a compound of formula I-C:

wherein G is —F, —Cl, —Br, —I, —N₃, —OR, SR, —ONRR, RC(═O)O—, orRSO₂—O—; and R is optionally substituted alkyl, or optionallysubstituted aryl.
 20. The method of claim 16, wherein conjugating thesecond moiety to the cysteine side chains of A comprises reacting acompound of formula I-D with a compound of formula I-E to provide acompound of formula I-F:

wherein each X is —Cl, —Br, —I, or RSO₂—O—; and R is optionallysubstituted alkyl, or optionally substituted aryl.
 21. The method ofclaim 20, wherein the cysteine side chains were reduced with a reducingagent to prepare compound I-D.
 22. The method of claim 16, whereinconjugating the second moiety to the cysteine side chains of A comprisesreacting a compound of formula II-A with a compound of formula I-E toprovide a compound of formula II-B:

wherein each X is —Cl, —Br, —I, or RSO₂—O—; and R is optionallysubstituted alkyl, or optionally substituted aryl.
 23. The method ofclaim 16, wherein conjugating the first moiety to the lysine side chainof A comprises reacting a compound of formula II-A with a compound offormula I-B to provide a compound of formula I-F:

wherein G is —F, —Cl, —Br, —I, —N₃, —OR, SR, —ONRR, RC(═O)O—, orRSO₂—O—; and R is optionally substituted alkyl, or optionallysubstituted aryl.
 24. The method of claim 16, wherein:

is:


25. The method of claim 16, wherein L¹ comprises

where X¹ is N (nitrogen) or CH; Y¹ is N (nitrogen), or CH; and p is 0,1, or
 2. 26. The method of claim 16, wherein L1 comprises


27. The method of claim 16, wherein D¹ is a tubulin binder, DNAalkylator, DNA intercalator, enzyme inhibitor, immune modulator,peptide, or nucleotide.
 28. The method of claim 16, wherein D² is atubulin binder, DNA alkylator, DNA intercalator, enzyme inhibitor,immune modulator, peptide, or nucleotide.
 29. The method of claim 16,wherein L1 or L² comprises —(CH₂)_(n)— where n is 1, 2, 3, 4, 5, 6, 7,8, 9, or
 10. 30. The method of claim 16, wherein L1 or L² includes—(CH₂CH₂O)_(n)— where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or
 10. 31. Themethod of claim 16, wherein the S-linked portion of

comprises a modified L-Alanine residue.
 32. The method of claim 16,wherein each

is:

wherein G is —NH—.
 33. The method of claim 16, wherein each

is:


34. The method of claim 16, wherein the dual-drug conjugate is:

or a pharmaceutically acceptable salt thereof, wherein A comprises anantibody or antigen-binding antibody fragment.
 35. The method of claim16, wherein A comprises an antibody.
 36. The method of claim 16, whereinA comprises an antigen-binding antibody fragment.